Ischemic stroke and the following reperfusion, an acute therapeutic intervention, can cause irreversible brain damages. However, the underlying pathological mechanisms are still under investigation. To obtain a comprehensive, real-time view of the cell-autonomous mechanisms involved in ischemic stroke and reperfusion, we applied the next-generation sequencing (NGS) technology to characterize the temporal changes in gene expression profiles using primarily cultured hippocampal neurons under an oxygen-glucose deprivation/reperfusion (OGD/R) condition. We first identified the differentially expressed genes (DEGs) between normal cultured neurons, neurons with OGD, and neurons with OGD followed by reperfusion for 6 h, 12 h, and 18 h, respectively. We then performed bioinformatics analyses, including gene ontological (GO) and pathway analysis and co-expression network analysis to screen for novel key pathways and genes involved in the pathology of OGD/R. After we confirmed the changes of selected key genes in hippocampal cultures with OGD/R, we further validated their expression changes in an in vivo ischemic stroke model (MCAO). Finally, we demonstrated that prevention of the up-regulation of a key gene (Itga5) associated with OGD/R promoted hippocampal neuronal survival. Our research thereby provided novel insights into the molecular mechanisms in ischemic stroke pathophysiology and potential targets for therapeutic intervention after ischemic stroke.